The invention relates generally to the field of cellular and subcellular analysis and more particularly, but not by way of limitation, to high throughput imaging devices and systems for use in cellular, sub-cellular and tissue analysis.
Traditional microscopy includes well-known techniques to view and image tissues, cells and subcellular structures. Using one or more light sources (sequentially or in combination) to obtain bright field, dark field, fluorescent, phase contrast or polarization information, image data can be collected by moving a microscope slide, or other cellular container, into the path of a stationary light source and objective. Moving the slide or sample container is typically a slow operation (e.g., 40 micron steps) to accommodate a limited field-of-view (“FOV”). The resolving limit of a light microscope is approximately 0.2 micron (often refereed to as wavelength limited), thus requiring approximately ±0.05 micron of translation resolution in the plane of the viewing field (hereinafter referred to as the horizontal (“x”) and vertical (“y”) axes). The high-resolution requirements for the component moving the sample container combined with the large area of a typical microscope slide (400 micron2 to 800 micron2), impose sever limits on transnational speeds. In these instruments, x-y translation is further encumbered by the time lost due to deceleration, acceleration, and backlash compensation of the moving component between each step or movement.
One current attempt to increase the analysis throughput of microscopy-based devices involves the analysis of quantities of reagents or analytes in the microliters using what are known as microwell plates or arrays. Existing microwell technology uses 96, 384 and 1,536 microwell plates, wherein each well can retain between approximately 1 microliter and 1 milliliter of liquid. In instruments designed to accommodate microwell plates, a light source is placed beneath a cell and an objective above the cell such that a top-down cellular view is obtained. This approach is stressed in the literature because it tends to reduce any interposed refractive index changes caused by the microwell structure itself. While many standard objectives can compensate for the typical bottom thickness of a microwell (e.g., 170 microns), they exponentially lose resolving power as the thickness increases. For example, a millimeter thick microwell bottom can obscure subcellular detail using standard, uncompensated, objectives. Another recognized drawback to microscopes designed to view and image microwell-based samples is the difficulty of accurately aligning the well structure (most microwells have a curved bottom) to the axis of the objective. For example, to achieve wavelength limited resolution it is typically necessary to use an objective having a high magnification (≧60×) and a numerical aperture ≧0.7. This combination in a standard microscope objective can result in a short depth of field—on the order of 0.1 micron to 0.8 micron (the “z” axis). Accordingly, if the microwell bottom is tilted beyond 1 micron in the FOV, important data in the image plane can be out of focus, resulting in the loss of information.
Thus, it would be beneficial to provide a device and system that is capable of high throughput imaging of multiple samples at a high resolution and that overcomes the acknowledged drawbacks to existing imaging systems.